Portions of the information set forth herein have been published. See W. L. Cleveland et al., Nature 306(5929):56-57 (1983). Within this application several publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
This invention concerns a method for the direct production of monoclonal anti-idiotypic antibodies, preferably ones that mimic receptors or ligands of receptors. A receptor is defined herein as a molecular structure that interacts with another structure, referred to as a ligand, as part of a biological process. Receptors can include, but are not restricted to, enzymes, immunoglobulins, lymphokines, cell surface molecules, attachment sites on viruses and cells, specific binding proteins such as those which bind nucleic acids, hormone binding molecules and metal-binding molecules such as calmodulin. A ligand is similarly defined as a structure that reacts with a receptor as defined above. In addition to binding to a receptor, a ligand as defined herein may act as an agonist or as an antagonist with respect to the receptor.
Antibodies arise when an animal is immunized with a particular antigen. The variable regions of such antibodies contain a set of antigenic determinants known as the idiotype which is usually associated with antigen specificity. Anti-idiotypic antibodies may arise when an animal is injected with specific idiotypic antibody molecules which have been previously obtained and purified. In such an experiment the animal immunized with the idiotypic antibody produces antibodies directed against the idiotypic determinants of the injected antibody. The idiotypic antibodies may then bind to either the antigen or the anti-idiotypic antibodies so produced. Regardless of functional differences, macromolecules having the same binding specificities can also show homologies at their binding sites. Thus immunizing an animal with purified idiotypic antibodies raised against an antigen which is a ligand for a biological receptor may raise anti-idiotypic antibodies which bind to both the idiotypic antibodies and the receptor for the ligand (1).
Anti-idiotype antibodies thus afford one route to functional anti-receptor antibodies, which have been implicated in several auto-immune diseases. At least some of these diseases (1,2) might originate from an antiidiotypic response to antibodies formed against biologically active ligands normally present in vivo, such as insulin or thyrotropin. The likelihood of an antiidiotypic etiology increases if the patients' antibodies are found to be directed at the combining site of a receptor Although not always the case (3), this has been found to be true in many patients with myasthenia gravis (4), particularly in those who are severely ill. Moreover, it has been shown (1) that experimental myasthenia gravis can be induced in rabbits via the antiidiotypic route. Thus, an aberrant anti-idiotypic response could have a role in at least some cases of myasthenia gravis in humans. In Graves disease, the specificity of circulating anti-thyroid receptor antibodies is usually directed at the combining site of the thyrotropin receptor (5). These anti-thyroid receptor antibodies are therefore probably anti-idiotypic, directed at idiotypes of anti-thyrotropin antibody.
Adenosine receptors are also important for several reasons. They are involved in the regulation of blood flow in arteries and arterioles, in particular in the heart. Thus, they are one of the many factors involved in determining blood circulation in this organ. They are also involved in the modulation of nervous impulses and, generally speaking, tend, when activated, to have a calming effect on animals. There is good evidence that these receptors are the targets for caffeine, which seems to reverse the action of adenosine and produce agitation, rather than tranquility. Finally, there is recent evidence that alcoholism may very well be associated with abnormalities in the adenosine receptor.
Previous methods for the production and study of anti-receptor antibodies required immunizing animals with purified receptors in order to raise the desired anti-bodies (6,7,8).
Recently (1), a procedure was described for preparing antibodies to the acetylcholine receptor (AChR) based on immunoglobulin idiotypes and on the above-mentioned hypothesis that, regardless of functional differences, macromolecules of the same specificity will show structural homologies in their binding sites. Antibodies were prepared in rabbits to a structurally constrained agonist of AChR, trans-3,3'-bis( -trimethylammonium) azotoluene bromide (BisQ) (9,10). These antibodies mimicked the binding specificity of AChR in its activated state (11) i.e., agonists were bound with affinities that were in accord with their biological activities while antagonists were bound poorly. Rabbits were then immunized with a specifically purified preparation of anti-BisQ antibodies to elicit a population of antiidiotypic antibodies specific for the binding sites of anti-BisQ. A portion of the anti-idiotypic antibodies (12,13) produced in the second set of rabbits crossreacted with determinants on AChR preparations from Torpedo californica, Electrophorus electricus and rat muscle. Moreover, several of the rabbits showed signs of experimental myasthenia gravis, in which circulating AChR antibodies are typically found.
Anti-idiotypic antibodies against the thyrotropin receptor have also been reported (14). In that experiment, thyrotropin (TSH) specific antibodies (idiotypic) raised in rats were injected into rabbits which then produced the anti-idiotypic antibodies against the rat anti-TSH antibodies.
It has been postulated that the anti-idiotypic response plays a role in regulating the immune response (12,13,15). According to this theory, injection of an antigen elicits, in addition to antibodies to the antigen, other populations that include anti-idiotypic antibodies directed at the combining sites of the antigen-specific antibodies. If the antigen-specific antibodies recognize a ligand of a receptor, then the antiidiotypic antibodies should bind receptor.
In the past, the spontaneous generation of anti-idiotypes in response to immunization against an antigen has seldom been detected (2). Recently however, the spontaneous appearance of auto-anti-idiotypic antibody was observed during a normal human immune response to tetanus toxoid (16). Similarly, immunization with insulin was observed to cause the spontaneous appearance of insulin receptor-specific antibodies (2).
The difficulty in detecting the anti-idiotypic response results from the low titres of circulating anti-idiotypic antibodies formed (1,2) and to the observation that the cellular events giving rise to the anti-idiotypic response are only ,a transient phenomenon. Attempts to utilize the auto-anti-idiotypic response to produce anti-idiotypic antibodies would also involve technical problems that arise from the formation of immune complexes, an important limitation of serological studies.
The present invention surprisingly overcomes the aforementioned limitations by providing a novel method for producing anti-idiotype antibodies which depends on the use of hybridoma technology to immortalize the cells which produce the anti-idiotype antibodies. By immortalizing and cloning the cells active at the time of cell harvest, the cellular events are "frozen" in time, making it possible to produce and study clones and large quantities of their products that may have only a transient existence in vivo. Moreover, since the immortalized clones are separated from each other, this approach avoids the technical problems that arise from the formation of immune complexes. It should be understood that the method of this invention eliminates the need to obtain a purified idiotypic antibody in order to produce the anti-idiotypic antibody and the need to obtain a purified receptor in order to produce an antireceptor antibody.
In view of the low efficiency usually observed for cell fusion (about one antibody-producing cell in one thousand is immortalized), combined with the low titres of naturally occurring anti-idiotypic antibodies and the possibility that the low titres may well have resulted from suppression of the very cells that produce such antibodies, it should further be understood that the success of this auto-anti-idiotypic method is indeed an unexpected and surprising result.